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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 23, 2024 |
| Title |
HEK293T, WT, groseq, Rep2 |
| Sample type |
SRA |
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| Source name |
kidney
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| Organism |
Homo sapiens |
| Characteristics |
tissue: kidney
cell line: HEK293T
cell type: human embryonic kidney
genotype: wild type
fraction: nascent RNA
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| Treatment protocol |
LN-229 cells were transfected in triplicate with 100nM of siRNA oligonucleotides targeting human ZFX (Horizon #L006572000005) or control oligonucleotides (Horizon #D0018101005) using Lipofectamine RNAiMAX (Thermo Fisher #13778150) for 72 hours.
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| Growth protocol |
LN-229 and HEK293T were grown using DMEM supplemented with 10% FBS and pen/strep.
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| Extracted molecule |
total RNA |
| Extraction protocol |
5x106 cells per nuclear run-on reaction were harvested and incubated in swelling buffer (10 µM Tris-HCl, 2 mM MgCl2, 3 mM CaCl2). Nuclei were isolated with lysis buffer (10 µM Tris-HCl, 2 mM MgCl2, 3 mM CaCl2, 10% glycerol, 1% NP-40). Nuclear run-on was performed at 30°C for 5 min in 10 mM Tris-HCl pH 8, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 500 µM ATP, 500 µM GTP, 500 µM Br-UTP, 2 µM CTP, 200 U/mL Superase In RNase Inhibitor (Invitrogen #AM2696), and 1% sarkosyl. Nuclear RNA was isolated with Trizol (Invitrogen #15596026). RNA was purified with Micro Bio-Spin P-30 Gel Columns (Bio-Rad # 7326250), fragmented with RNA Fragmentation Kit (Invitrogen # AM8740), and treated with 15 units mRNA decapping enzyme (NEB # M0608S) and 30 units T4 PNK (NEB # M0201S). RNA immunoprecipitation was performed 3 times with anti-BrdU-conjugated agarose beads (Santa Cruz Biotechnologies # sc-32323 AC).
Library preparation was performed with TruSeq Small RNA Library Preparation Kit (Illumina # RS-200-0012 following the manufacturer’s protocol.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads with Illumina small RNA adapter sequences were trimmed using Trimgalore
Reads were aligned to hg38 using Tophat
Normalization was done across samples using an equal number of uniquely mapped reads
Assembly: hg38
Supplementary files format and content: bigwig file with total mapped reads
Library strategy: GRO-seq
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| Submission date |
Aug 21, 2023 |
| Last update date |
Apr 23, 2024 |
| Contact name |
Peggy J Farnham |
| E-mail(s) |
peggy.farnham@med.usc.edu
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| Organization name |
University of Southern California
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| Department |
Biochemistry and Molecular Medicine
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| Street address |
1450 Biggy St, NRT6514
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE241281 |
Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters [GRO-seq] |
| GSE241284 |
Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters |
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| Relations |
| BioSample |
SAMN37106833 |
| SRA |
SRX21447433 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7720587_USC_GRO003_293t_groseq_rep2_combined_runs_minus.bigwig |
147.7 Mb |
(ftp)(http) |
BIGWIG |
| GSM7720587_USC_GRO003_293t_groseq_rep2_combined_runs_plus.bigwig |
149.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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